A new in vitro test for pyrimethamine/sulfadoxine susceptibility of Plasmodium falciparum and its correlation with in vivo resistance in Kenya

Bulletin ofthe WorldHealth Organization, 62 (4): 615- 621 (1984) © World Health Organization 1984 A new in vitro test for pyrimethamine/sulfadoxine susceptibility of Plasmodium falciarum and its correlation with in vivo resistance in Kenya H. C. SPENCER,1 W. M. WATKINS,2 D. G. SIXSMITH,2 D. K. KOECH,3 & J. D. CHULAY4 A useful in vitro methodforfield evaluation ofPlasmodium falciparum sensitivity to pyrimethamine/sulfadoxine is described. Thirty-five Kenyan schoolchildren infected with P. falciparum were treated with this drug combination andfollowed upforS weeks. In vitro testsfor sensitivity to these drugs and to chloroquine wereperformed before starting treat- ment. All infections cleared within 7 days oftreatment, but 5 children had recurrent parasi- taemia within 35 days. The original isolates from 4 of these S children had an in vitro response topyrimethamine/sulfadoxine similar to a known strain that was resistant to these drugs; only 4 ofthe remaining 30 isolatesfrom patients in whom recurrentparasitaemia did not occur had a resistant in vitro response (P = 0.006). In thepatient with recurrent parasi- taemia whose initial isolate appearedsensitive topyrimethamine/sulfadoxine, the recurrent isolate had a resistant pattern in vitro, suggesting either reinfection or selection of a resistant subpopulationfollowing treatment. The in vitro response to this drug combination was correlated with the in vitro response to either drug alone and with the in vitro response to chloroquine. Two of the 5 infections with recurrent parasitaemia after initial pyri- methamine/sulfadoxine treatment were resistant to chloroquine in vivo. The in vitro test for pyrimethamine/sulfadoxine should be useful for mapping the spread of multidrug- resistant P. falciparum. The emergence in East Africa of Plasmodium fal- ciparum infections resistant to chloroquine has resulted in the increased use of the antimalarial drug combination of pyrimethamine/sulfadoxine (Fansidar) (1-2). However, falciparum malaria resis- tant to this drug combination has been reported from many places (3-9) and simultaneous resistance to chloroquine and pyrimethamine/sulfadoxine has occurred occasionally in East Africa (7-9). The rapidly changing drug response of P.falciparum infections in East Africa warrants continued surveil- lance of drug sensitivity patterns. For the monitoring of drug sensitivity patterns it is essential to have a simple in vitro test for field evalua- tion of P.falciparum drug sensitivity. To date there has been no suitable test for pyrimethamine/sulfa- doxine sensitivity. The response of P.falciparum to pyrimethamine (but not to sulfadoxine) can be ' Malaria Coordinator, Clinical Research Centre, Kenya Medical Research Institute, Nairobi, Kenya; and Medical Epidemiologist, Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333, USA. Requests for reprints should be sent to this author at the Atlanta address. 2 Senior Lecturers, Department of Pharmacy, University of Nairobi, Nairobi, Kenya. 3 Director, Clinical Research Centre, Kenya Medical Research Institute, Nairobi, and Head, Division of Vector-borne Diseases, Ministry of Health, Nairobi, Kenya. 4 Medical Officer, US Army Medical Research Unit, Kenya. measured by using standard culture techniques with RPMI medium 1640 (10); this medium contains high concentrations of para-aminobenzoic acid (PABA) and folic acid, which antagonize the inhibitory effects of sulfadoxine (11). An in vitro system for evaluation of synergy between pyrimethamine and sulfadoxine against P.falciparum was recently developed in our labora- tories (11). Using modified RPMI medium 1640, with no PABA but containing folic acid equivalent to a physiological plasma folate concentration, synergy between pyrimethamine and sulfadoxine was demon- strated for pyrimethamine-sensitive and -resistant strains of P.falciparum. In this paper we present the results of a field evaluation of this in vitro test for pyrimethamine/sulfadoxine sensitivity in Kenya, with comparative data on the in vivo response to pyri- methamine/sulfadoxine treatment and on chloro- quine sensitivity. MATERIALS AND METHODS Study population The investigation was conducted during January-March 1983 in Malindi, Kenya, where 4438 -615 616 H. C. SPENCER ET AL. malaria is hyperendemic. Schoolchilden with pure P.falciparum infections were identified in surveys and included in the study if they were not acutely ill, had at least 50 asexual parasites per 300 white blood cells (WBC) (approximately 1000 per ul), had a negative urine sample for 4-aminoquinolines using the Dill-Glazko technique (12) and for free sulfa using a modification of the Bratton-Marshall method (13), gave no history of drug ingestion during the pre- vious 2 weeks, were old enough for venepuncture to be performed, and had permission to participate given by their teachers. Children with malaria infec- tions not included in the study were treated with chloroquine phosphate according to Government of Kenya regulations. In vivo tests A 35-day in vivo test for sensitivity to pyri- methamine/sulfadoxine, similar to the extended WHO test for chloroquine (14), was performed on each child. The children were weighed and treated on day 0 with tablets, each containing 25 mg pyrimetha- mine and 500 mg sulfadoxine, according to their body weight: < 25 kg body weight = 1 tablet; 25- 37.5 kg = 11 tablets; 37.6-50 kg = 2 tablets. Treat- ment was given in the presence of one of the investi- gators and each child was observed for at least 30 minutes to ensure that vomiting did not occur. Thick blood films were obtained daily through day 7 and weekly thereafter through day 35. Asexual parasites were counted per 300 WBC; the entire smear was examined for negative slides. Urine samples were tested for 4-aminoquinolines daily through day 7 and for sulfa daily until positive. Patients were excluded from the study if they developed a positive Dill- Glazko test, which suggests chloroquine ingestion, or if the urine sulfa test did not become positive within 7 days, which suggests failure to absorb the drug. Patients in whom P.falciparum parasitaemia recurred during the follow-up period were treated with chloroquine, 25 mg of base/kg of body weight over 3 days. The drug was administered by technical staff and the patient observed for 30 minutes to ensure that vomiting did not occur. Blood smears were taken daily through day 7 (4 cases) or only on days 6 and 7 (1 case). In vitro tests Before treatment and at the time of recurrent para- sitaemia, 5-10 ml blood was collected by vene- puncture and transferred to a sterile tube containing 143 units of heparin. Pyrimethamine/sulfadoxine test. Flat-bottomed, 96-well microculture plates were predosed with pyri- methamine (Burroughs Wellcome, Research Triangle Park, NC),' sulfadoxine (Hoffmann-La Roche, Nutley, NJ) or both. The drugs were dissolved in 70% ethanol, serial dilutions were made with ethanol, and 20 1l aliquots were added to appropriate wells. The plates were incubated at 37 °C for 24 hours to allow evaporation and stored at 4 °C until used (2-4 weeks later). One test utilized 32 wells at the following final concentrations: 2 controls with no drug; pyri- methamine alone (0.0001, 0.001, 0.003, 0.01, 0.03, 0. 1, 0.3, 1, 2, 3, 4, 8, 16 and 32 Armol/l); the same con- centrations of pyrimethamine plus sulfadoxine (3.22 ,gmol/l); and sulfadoxine alone (32.2 and 322 umol/l). In the field laboratory, 0.3 ml of para- sitized blood was added to 7.2 ml of modified RPMI medium 1640 (with no PABA, and folic acid at 22.7 nmol/l) supplemented with HEPES (25 mmol/l), NaHCO3 (25 mmol/l), gentamicin (10 mg/I), and 10% normal human AB (+) serum. Aliquots (0.2 ml) were added to the wells of the predosed plates, which were then placed in a gastight box, flushed with a gas mixture containing 3% CO2, 5% 02 and 92% N2, and incubated at 37 'C. After 48 hours the supernatant fluid was removed from each well and a thin blood film was made from the remaining erythrocyte suspension. Slides were stained with Giemsa and the number and stage of development of parasites in at least 5000 erythrocytes were counted. A test was con- sidered successful if the control wells without drug had at least a 2-fold increase in total parasitaemia compared with the count prior to incubation. The minimum inhibitory concentration (MIC) was the lowest concentration at which no ring forms were found. Chloroquine test. A modified 48-hour test for chloroquine sensitivity was performed as previously described (15), with minor modifications. This test was essentially the same as the pyrimethamine/sulfa- doxine test, with the following exceptions: normal RPMI medium 1640, supplemented as above, was used to dilute the parasitized blood; 0.5 ml aliquots were added to the wells of a 24-well tissue culture plate containing chloroquine phosphate (Dawa Pharma- ceuticals, Nairobi, Kenya) to give final concen- trations of 0 (control), 0.01, 0.03, 0.06, 0.1 and 0.3 tmol/l; and the MIC was the lowest concentration at which net multiplication did not occur. Known chloroquine-sensitive isolates have an MIC of < 0.03 /tmol/l (15, 16). In vitro tests on laboratory isolates. Two reference laboratory strains were tested for in vitro sensitivity to pyrimethamine/sulfadoxine by Dr Phuc Nguyen- Dinh (Malaria Branch, Division for Parasitic Diseases, Centers for Disease Control), using plates a Use of brand names and commercial sources is for clarifica- tion and does not imply endorsement by the US Public Health Service or the Government of Kenya. PYRIMETHAMINE/SULFADOXINE SUSCEPTIBILITY OF PLASMODIUM FALCIPARUM and media supplied by our laboratory. The Indochina I strain (pyrimethamine/sulfadoxine-resistant) was isolated from a patient with recurrent parasitaemia after treatment with pyrimethamine/sulfadiazine (17). The P143 strain (pyrimethamine/sulfadoxine- sensitive) was isolated from a patient in Haiti whose infection was cured after treatment with pyrimeth- amine/sulfadoxine with no recrudescence during a 28-day follow-up period (Dr Phuc Nguyen-Dinh, personal communication). Drug levels Blood and urine specimens were collected on days 0 and 2. Samples from patients with recurrent para- sitaemia were examined for pyrimethamine by Dr Fred Churchill (Division of Parasitic Diseases, Centers for Disease Control) using high pressure liquid chromatography with UV absorbance detector at 290 nm. Statistical analysis Fisher's exact test was used for 2 x 2 contingency tables, and a regression coefficient was calculated where appropriate; P < 0.05 was considered signifi- cant. RESULTS Study population The initial study group consisted of 41 children. Six children were excluded because one developed a posi- tive Dill-Glazko test on day 2, one was lost to follow- up after day 4, one failed to develop a positive urine sulfa test despite repeated testing through day 7, and the pyrimethamine/sulfadoxine in vitro test was un- successful in three. The final group consisted of 24 boys and 11 girls, aged 6-15 years. In vivo tests The parasitaemia cleared in 3 children on day 1, in 31 on day 2, and in 1 child each on days 3 and 4. No parasites were detected on days 4, 5 or 7. Two children had no parasites detected on days 4 or 5, a few para- sites on day 6, and none on day 7. Mean parasite counts were 274 (range, 55-1131) asexual parasites per 300 WBC on day 0,57 (range, 0-314) on day 1, 0.1 (range, 0-3) on days 2 and 6, and 0.03 (range, 0- 1) on day 3. During the 35-day follow-up period, 5 children developed recurrent parasitaemia: 1 on day 21 (para- sitaemia of 114 asexual parasites per 300 WBC), 1 on day 28 (9 parasites per 300 WBC), and 3 on day 35 (10, 33 and 780 parasites per 300 WBC). Drug levels Blood and urine specimens collected on days 0 and 2 from the patients with recurrent parasitaemia were examined for pyrimethamine. Pyrimethamine could not be detected (< 0.08 Amol/l) in the day 0 specimens. On day 2, the pyrimethamine levels in the blood ranged between 0.63 and 1.03 (mean = 0.87) Amol/l and in the urine between 0.71 and 5.87 (mean = 2.95) ztmol/l. In vitro tests The pyrimethamine/sulfadoxine-sensitive P143 strain and the pyrimethamine/sulfadoxine-resistant Indochina I strain had pyrimethamine/sulfadoxine MICs of 0.0001/3.22 Amol/l and 0.3/3.22 ,mol/l, respectively (Table 1). For the 35 field isolates the in vitro response to pyrimethamine/sulfadoxine was correlated with recurrent parasitaemia after pyrimethamine/sulfa- doxine treatment (Table 2). A pyrimethamine/sulfa- doxine MIC of > 0.1/3.22 zmol/l was predictive of recurrent parasitaemia. The in vitro response to pyri- methamine/sulfadoxine was also correlated with the in vitro response to either drug alone (Fig. 1, Table 3) and to chloroquine (Table 4). In vitro sensitivity testing was successful with two of the five P.falciparum isolates detected during the follow-up period. Three isolates from patients with low parasitaemia did not grow in culture. The isolates from patient M106 on days 0 and 21 had identical response patterns, but those from patient M150 on days 0 and 35 were quite different (Table 5). Table 1. In vitro responses to pyrimethamine, sulfadoxine, and pyrimethamine/sulfadoxine (P/S) of two reference Plasmodium falciparum strains Minimal inhibitory concentrations(jtmol/l) In vivo response Strain to P/S Pyrimethamine Sulfadoxine P/S P143 Sensitive 0.003 32.2 0.0001/3.22 Indochina Resistant 1 322 0.3/3.22 617 H. C. SPENCER ET AL. Table 2. Correlation0 between in vitro and in vivo responses to pyrimethamine/sulfadoxine in 35 Kenyan children with Plasmodium falciparum infections Minimal inhibitory concentrations (ymol/l) Recurrent Pyrimethamine/sulfadoxine parasitaemia after treatment S 0.03/3.22 > 0. 1/3.22 No 26 4 Yes 1 4 0 P = 0.006 (Fisher's exact test). Table 4. Correlation0 between in vitro sensitivities to chloroquine and pyrimethamine/sulfadoxine among 35 Plasmodium falciparum isolates from Malindi, Kenya. Minimal inhibitory concentrations (JtmoI/l) Pyrimethamine/sulfadoxine Chloroquine < 0.03/3.22 ) 0.1 /3.22 A0.1 20 2 >0.3 7 6 a P = 0.02 (Fisher's exact test). Table 3. Correlation' between in vitro sensitivities to sulfadoxine and pyrimethamine/sulfadoxine among 35 Plasmodium falciparum isolates from Malindi, Kenya. Minimal inhibitory concentrations (smoI/l) Pyrimethamine/sulfadoxine Sulfadoxine S 0.03/3.22 > 0.1/3.22 A322 24 3 >322 0 8 a P = 0.000007 (Fisher's exact test). f .3 _ e C . o 3: .1 -X s *0< 0 4 X .01 . C C5 e) #> .003 :. e (a 0 .001 -. E cs h. ..0001I Al. 0 0 0**/ *0 * 0 r = 0.75 0 0 00 .001 .003 .01 .03 .1 .3 1 4 Inhibitory Concentration of Pyrimethamine (Pi M) Fig. 1. Correlation between in vitro sensitivities to pyri- methamine and pyrimethamine/sulfadoxine among 35 Plasmodium falciparum isolates from Malindi, Kenya. The correlation was statistically significant (P < 0.01). Follow-up treatment The five patients with recurrent parasitaemia during the 35-day follow-up period were treated with chloroquine, 25 mg base/kg body weight over 3 days. In all five the initial isolates had been resistant to chloroquine in vitro (Table 5). Two recurrent infec- tions were resistant in vivo. Patient M150 had an RII pattern of chloroquine resistance (14) (reduction of parasitaemia but no clearance by day 7); he was referred to a local hospital for treatment with quinine. PatientM 106, who had 114 parasites per 300WBC on day 21 after pyrimethamine/sulfadoxine treatment, had 6, 7 and 42 parasites per 300 WBC on days 6, 7 and 8 after chloroquine treatment. Because blood films were not obtained on days 1-5, a distinction between RI and RII resistance could not be made. This patient responded to a second course of chloro- quine, the parasitaemia clearing within 4 days. The remaining three patients cleared their parasitaemia by day 7; since follow-up was not continued, resistance of the RI delayed recrudescence type was not excluded. DISCUSSION The described in vitro test for sensitivity of P.falciparum to pyrimethamine/sulfadoxine was easily performed under field conditions. Like cultured laboratory strains (11), the parasites ob- tained from infected patients readilv developed in the modified RPMI medium 1640 with no PABA and a physiological concentration of folate. Although we used chambers flooded with a defined gas mixture, we have successfully tested isolates from patients and from culture for sensitivity to pyrimethamine/sulfa- doxine with the same technique but using a candle jar to obtain a similar environment for growth. 618 PYRIMETHAMINE/SULFADOXINE SUSCEPTIBILITY OF PLASMODIUM FALCIPARUM Table 5. In vitro response to pyrimethamine, sulfadoxine, pyrimethamine/sulfadoxine (P/S), and chloroquine of Plasmodium falciparum isolates from five patients with recurrent parasitaemia after treatment with pyrimeth- amine/sulfadoxine. Minimal inhibitory concentrations (gmol/l) Patient Day of No. treatment Pyrimethamine Sulfadoxine P/S Chloroquine M130 0 1 > 322 0.3/3.22 >0.3 M123 0 1 > 322 0.1/3.22 0.3 M129 0 4 > 322 0.3/3.22 0.3 M106 0 1 > 322 0.1/3.22 > 0.3 M106 21 1 > 322 0.1/3.22 > 0.3 M150 0 0.003 32.2 0.001/3.22 > 0.3 M150 35 4 > 322 4/3.22 > 0.3 The in vitro test for sensitivity to pyrimethamine/ sulfadoxine was able to predict recurrent para- sitaemia. In 4 of the 5 infections in which para- sitaemia recurred during the 35-day follow-up period, the in vitro response to pyrimethamine/sulfadoxine was similar to that found for the drug-resistant laboratory strain. This suggests that these recurrences were due to recrudescence of a resistant parasite rather than reinfection. In the fifth case the initial isolate appeared sensitive in vitro, but the isolate obtained at the time of recurrent parasitaemia appeared resistant. These data suggest that reinfec- tion with a less sensitive isolate had occurred, or that a resistant subpopulation of parasites from the original infection had been selected following treat- ment with pyrimethamine/sulfadoxine. Host failure in treatment of malaria with pyri- methamine/sulfonamide combinations has been described as being due to poor bio-availability, abnormally rapid metabolism of sulfonamide, or other host factors (18-19). These are unlikely to explain our findings because of the in vitro evidence of drug resistance. Four children with an in vitro response predictive of resistance did not develop recurrent parasitaemia. Since all the children had lived in a malaria endemic area throughout their lives and had probably had repeated malaria infections, acquired partial immu- nity may have enhanced the response to drug treat- ment. Also, a longer follow-up period may have been needed to discover recrudescences. In non-immune volunteers infected with P.falciparum, recrudes- cences have occurred as long as 41-60 days after treatment with pyrimethamine/sulfonamide com- binations (20). The in vitro response to pyrimethamine/sulfa- doxine was correlated with that to pyrimethamine alone, as previously described (21), and to sulfa- doxine alone. Isolates less responsive to pyrimetha- mine alone and to sulfadoxine alone were generally less responsive in vitro to the combination. However, because some isolates were resistant to both drugs individually but sensitive to the combination, the in vitro response to pyrimethamine/sulfadoxine was better able to predict the in vivo response. The association between in vitro resistance to chloroquine and to pyrimethamine/sulfadoxine was unexpected. To our knowledge, treatment of malaria with pyrimethamine/sulfadoxine is uncommon in Malindi. It would appear that resistance to pyri- methamine/sulfadoxine exists in this area in the absence of drug pressure, or that resistance to pyri- methamine/sulfadoxine develops simultaneously with resistance to chloroquine. Alternatively, multi- drug-resistant strains of P.falciparum may have been introduced from south-east Asia. Our data suggest that pyrimethamine/sulfadoxine may quickly become ineffective for treating malaria in Malindi. Previous work in this community has demonstrated that more than 75070 of P.falciparum infections are resistant to pyrimethamine in vivo and irn vitro (Spencer et al., unpublished results). Since 1980, surveys have also demonstrated increasing resis- tance to chloroquine in Malindi (22). In Thailand, resistance of P.falciparum to chloroquine (23) developed prior to resistance to pyrimetha- mine/sulfadoxine (24); only recently has simul- taneous resistance to chloroquine and pyrimetha- mine/sulfadoxine been observed (6). Continued sur- veillance of antimalarial drug sensitivity should be continued in Kenya and elsewhere. The described in vitro test for pyrimethamine/sulfadoxine sensitivity sho. ild be a useful tool for mapping the prevalence of resistance to this antimalarial combination. 619 620 H. C. SPENCER ET AL. ACKNOWLEDGEMENTS We thank the Director of Medical Services, Kenya Ministry of Health, and the Director, Kenya Medical Research Institute,for permission to publish this paper. We aregrateful for the excellent technical assistance of David Boriga Anyona, Daniel M. Kariuki, Joseph Gitau and Robert Nzovu of the Division of Vector-borne Diseases, Kenya Ministry of Health. This investigation received support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and from the Kenyan National Council for Science and Technology. RtSUMt NOUVELLE EPREUVE IN VITRO DE DETERMINATION DE LA SENSIBILITE DE PLASMODIUMFALCIPARUM A LA SULFADOXINE/PYRIMETHAMINE ET CORRELATION AVEC LA RESISTANCE IN VIVO AU KENYA Les auteurs decrivent une methode in vitro utile pour l'evaluation sur le terrain de la sensibilite de Plasmodium fakciparum A la sulfadoxine/pyrimethamine. Trente-cinq cas d'infestation d'ecoliers kenyens par P.falciparum ont e traites avec cette association medicamenteuse et suivis pendant 5 semaines. La sensibilite A ces drogues ainsi qu'A la chloroquine a e determin6e in vitro avant la mise en route du traitement. Les signes d'infestation ont disparu dans tous les cas dans les 7 jours qui ont suivi le debut du traitement mais une recidive de parasitemie a e observee chez 5 des enfants dans les 35 jours suivants. Chez 4 de ces 5 enfants, on a note une reaction in vitro des isolements initiaux A la sulfadoxine/pyrimethamine semblable A celle d'une souche connue pour etre resistante A ces medicaments; parmi les 30 autres isolements (correspondant aux cas sans recidive) 4 seulement ont manifeste une resistance in vitro (P = 0,006). L'isolement effectue pendant la recidive de parasitemie chez le patient dont l'isolement initial s'etait montre sensible a la sulfadoxine/pyrimethamine a manifeste une resistance in vitro, donnant a penser qu'il y avait eu soit reinfestation soit selection d'une sous-population resistante a la suite du traitement. La reaction in vitro a cette association m6dica- menteuse etait correlee avec les reactions in vitro a chacun des deux medicaments associes et a la chloroquine. Une resistance in vivo a la chloroquine a e observee chez 2 des 5 cas de recidive de parasitemie apres le traitement initial par la sulfadoxine/pyrimethamine. 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